The posttranslational attachment of oligosaccharides is required for proper folding and transport of many secreted and integral membrane glycoproteins. Human CD4, the 55kD protein present on a subset of T lymphocytes and the receptor for the human immunodeficiency virus (HIV), contains two potential asparagine-linked glycosylation sites within its extracellular domain. Previous work has suggested that glycosylation is required for cell surface expression of CD4 and that unglycosylated CD4 is unstable and rapidly degraded. Other studies have shown that unglycosylated CD4 can function as a cell surface receptor for HIV. To clarify and further define the role of glycosylation we generated a series of mutant cDNAs in which one, the other, or both potential glycosylation recognition sites were eliminated. Using in vitro transcription and translation we confirmed that both potential glycosylation sites of CD4 are utilized. We transiently expressed the mutant cDNAs in human cells and precipitated the resulting proteins using monoclonal and polyclonal antibodies to CD4. Using this approach we demonstrated that glycosylation at either site was necessary and sufficient for proper protein folding and transport to the cell surface. We have found that unglycosylated CD4 is synthesized in the cell at levels comparable to wild type CD4 and can be precipitated only by the polyclonal antiserum, suggesting that unglycosylated CD4 is improperly folded and retained intracellularly, probably in the endoplasmic reticulum (ER).